Preliminary work has produced a surprising, and potentially important finding for understanding the etiology of Rett Syndrome. The new data show that simply by binding to chromatin, MeCP2 induces a dramatic change in 3D conformation, resulting in a marked increase in compaction. Unlike the chromatin compaction induced by histone H1, which is highly dependent on salt concentration and produces a zig-zag conformation, MeCP2 compaction is independent of salt concentration, and proceeds through edge-to-edge contact between nucleosomes. Further, this MeCP2-induced compaction appears not to require DNA methylation. Work by others has clearly demonstrated that the gene silencing function of MeCP2 can occur by the recruitment of histone deacetylase (HDAC) complexes, followed by hypoacetylation of core histones leading to the generation of local areas of heterochromatin. The new findings suggest a dual mechanism of heterochromatin formation by MeCP2 - one component being an intrinsic consequence of MeCP2 binding to chromatin, the other via HDAC recruitment. This proposal is aimed at understanding the molecular mechanism(s) by which MeCP2 compacts chromatin, and the impact on this mechanism of MeCP2 mutants known to be important in RTT. A primary technique to be used in the proposed research is direct imaging by electron microscopy (EM), complemented by appropriate biochemical and biophysical tools. EM has the advantage over 'bulk' techniques that the 3D conformation of chromatin is accessible, allowing the locations and orientations of nucleosomes in individual chromatin arrays to be found. Specific aims include: determining which domain(s) of MeCP2 contribute to its ability to compact chromatin; determining the relative contributions of nucleosomal and linker DNA in MeCP2 binding; and, in view of the report that MeCP2 can displace histone H1 from chromatin, determining whether H1 and MeCP2 share the same or overlapping binding sites on chromatin. The results will provide fundamental new information of the interactions of MeCP2 with chromatin, information that can be drawn on as potential therapies for RTT are formulated and evaluated.